Effects of cGMP and sodium nitroprusside on odor responses in turtle olfactory sensory neurons

The effects ofcGMP and sodium nitroprusside (SNP) on odor responses in isolatedturtle olfactory neurons were examined. The inward current induced bydialysis of a mixture of 1 mM cAMP and 1 mM cGMP was similar to thatinduced by dialysis of 1 mM cAMP or 1 mM cGMP alone. After the neuronswere desensitized by the application of 1 mM cGMP, 3 mM8-(4-chlorophenylthio)-cAMP, a membrane-permeable cAMP analog, did notelicit any current, indicating that both cAMP and cGMP activated thesame channel. Extracellular application of SNP, a nitric oxide (NO)donor, evoked inward currents in a dose-dependent manner. However,application of SNP did not induce any currents after desensitization ofthe cGMP-induced currents, suggesting that SNP-induced currents aremediated via the cGMP-dependent pathway. Application of thecAMP-producing odorants to the neurons induced a large inward currenteven after neurons were desensitized to a high concentration of cGMP orSNP. These results suggest that the transduction pathway independent ofcAMP, cGMP, and NO also contributes to the generation of odor responsesin addition to the cAMP-dependent pathway.

     

Regulation of C. elegans longevity by specific gustatory and olfactory neurons

The life span of C. elegans is extended by mutations that inhibit the function of sensory neurons. In this study, we show that specific subsets of sensory neurons influence longevity. We find that certain gustatory neurons inhibit longevity, whereas others promote longevity, most likely by influencing insulin/IGF-1 signaling. Olfactory neurons also influence life span, and they act in a distinct pathway that involves the reproductive system. In addition, we find that a putative chemosensory G protein-coupled receptor that is expressed in some of these sensory neurons inhibits longevity. Together our findings imply that the life span of C. elegans is regulated by environmental cues and that these cues are perceived and integrated in a complex and sophisticated fashion by specific chemosensory neurons.     

Ethanol preference in C. elegans

Caenorhabditis elegans senses multiple environmental stimuli through sensory systems and rapidly changes its behaviors for survival. With a simple and well-characterized nervous system, C. elegans is a suitable animal model for studying behavioral plasticity.
Previous studies have shown acute neurodepressive effects of ethanol on multiple behaviors of C. elegans similar to the effect of ethanol on other organisms. Caenorhabditis elegans also develops ethanol tolerance during continuous exposure to ethanol. In mammals, chronic ethanol exposure leads to ethanol tolerance as well as increased ethanol consumption. Ethanol preference is associated with the development of tolerance and may lead to the development of ethanol dependence.
In this study, we show that C. elegans is a useful model organism for studying chronic effects of ethanol, including the development of ethanol preference. We designed a behavioral assay for testing ethanol preference after prolonged ethanol exposure. Despite baseline aversive responses to ethanol, animals show ethanol preference after 4 h of pre-exposure to ethanol and exhibit significantly enhanced preference for ethanol after a lifetime of ethanol exposure. The cat-2 and tph-1 mutant animals have defects in the synthetic enzymes for dopamine and serotonin, respectively. These mutants are deficient in the development of ethanol preference, indicating that dopamine and serotonin are required for this form of behavioral plasticity.     

The claudin superfamily protein nsy-4 biases lateral signaling to generate left-right asymmetry in C. elegans olfactory neurons

Early in C. elegans development, signaling between bilaterally symmetric AWC olfactory neurons causes them to express different odorant receptor genes. AWC left-right asymmetry is stochastic: in each animal, either the left or the right neuron randomly becomes AWC(ON), and the other neuron becomes AWC(OFF). Here we show that the nsy-4 gene coordinates the lateral signaling that diversifies AWC(ON) and AWC(OFF) neurons. nsy-4 mutants generate 2 AWC(OFF) neurons, as expected if communication between the AWC neurons is lost, whereas overexpression of nsy-4 results in 2 AWC(ON) neurons. nsy-4 encodes a transmembrane protein related to the gamma subunits of voltage-activated calcium channels and the claudin superfamily; it interacts genetically with calcium channels and antagonizes a calcium-to-MAP kinase cascade in the neuron that becomes AWC(ON). Genetic mosaic analysis indicates that nsy-4 functions both cell-autonomously and nonautonomously in signaling between AWC neurons, providing evidence for lateral signaling and feedback that coordinate asymmetric receptor choice.     

C. elegans: of neurons and genes

The human brain contains 100 billion neurons and probably one thousand times more synapses. Such a system can be analyzed at different complexity levels, from cognitive functions to molecular structure of ion channels. However, it remains extremely difficult to establish links between these different levels. An alternative strategy relies on the use of much simpler animals that can be easily manipulated. In 1974, S. Brenner introduced the nematode Caenorhabditis elegans as a model system. This worm has a simple nervous system that only contains 302 neurons and about 7,000 synapses. Forward genetic screens are powerful tools to identify genes required for specific neuron functions and behaviors. Moreover, studies of mutant phenotypes can identify the function of a protein in the nervous system. The data that have been obtained in C. elegans demonstrate a fascinating conservation of the molecular and cellular biology of the neuron between worms and mammals through more than 550 million years of evolution.     

High-throughput behavioral analysis in C. elegans

We designed a real-time computer vision system, the Multi-Worm Tracker (MWT), which can simultaneously quantify the behavior of dozens of Caenorhabditis elegans on a Petri plate at video rates. We examined three traditional behavioral paradigms using this system: spontaneous movement on food, where the behavior changes over tens of minutes; chemotaxis, where turning events must be detected accurately to determine strategy; and habituation of response to tap, where the response is stochastic and changes over time. In each case, manual analysis or automated single-worm tracking would be tedious and time-consuming, but the MWT system allowed rapid quantification of behavior with minimal human effort. Thus, this system will enable large-scale forward and reverse genetic screens for complex behaviors.     

Chemosensory signaling in C. elegans

The nematode Caenorhabditis elegans can sense and respond to hundreds of different chemicals with a simple nervous system, making it an excellent model for studies of chemosensation. The chemosensory neurons that mediate responses to different chemicals have been identified through laser ablation studies, providing a cellular context for chemosensory signaling. Genetic and molecular analyses indicate that chemosensation in nematodes involves G protein signaling pathways, as it does in vertebrates, but the receptors and G proteins involved belong to nematode-specific gene families. It is likely that about 500 different chemosensory receptors are used to detect the large spectrum of chemicals to which C. elegans responds, and one of these receptors has been matched with its odorant ligand. C. elegans olfactory responses are also subject to regulation based on experience, allowing the nematode to respond to a complex and changing chemical environment.     

A distributed chemosensory circuit for oxygen preference in C. elegans

The nematode Caenorhabditis elegans has complex, naturally variable behavioral responses to environmental oxygen, food, and other animals. C. elegans detects oxygen through soluble guanylate cyclase homologs (sGCs) and responds to it differently depending on the activity of the neuropeptide receptor NPR-1: npr-1(lf) and naturally isolated npr-1(215F) animals avoid high oxygen and aggregate in the presence of food; npr-1(215V) animals do not. We show here that hyperoxia avoidance integrates food with npr-1 activity through neuromodulation of a distributed oxygen-sensing network. Hyperoxia avoidance is stimulated by sGC-expressing oxygen-sensing neurons, nociceptive neurons, and ADF sensory neurons. In npr-1(215V) animals, the switch from weak aerotaxis on food to strong aerotaxis in its absence requires close regulation of the neurotransmitter serotonin in the ADF neurons; high levels of ADF serotonin promote hyperoxia avoidance. In npr-1(lf) animals, food regulation is masked by increased activity of the oxygen-sensing neurons. Hyperoxia avoidance is also regulated by the neuronal TGF-β homolog DAF-7, a secreted mediator of crowding and stress responses. DAF-7 inhibits serotonin synthesis in ADF, suggesting that ADF serotonin is a convergence point for regulation of hyperoxia avoidance. Coalitions of neurons that promote and repress hyperoxia avoidance generate a subtle and flexible response to environmental oxygen.     

Cellular and molecular analyses of olfactory behavior in C. elegans

C. elegans responds to and discriminates among a large number of volatile and water-soluble chemicals using a few defined chemosensory neurons. The functions of individual sensory neurons have been defined by cell killing experiments, and genes required for responses to subsets of chemicals have been identified. C. elegans has several large families of putative chemosensory receptor genes, and one of these genes has been demonstrated to encode a receptor for a specific odorant. Current work is aimed at identifying additional components of chemosensory neuron development and function.     

ROS and cGMP signaling modulate persistent escape from hypoxia in Caenorhabditis elegans

The ability to detect and respond to acute oxygen (O₂) shortages is indispensable to aerobic life. The molecular mechanisms and circuits underlying this capacity are poorly understood. Here, we characterize the behavioral responses of feeding Caenorhabditis elegans to approximately 1% O₂. Acute hypoxia triggers a bout of turning maneuvers followed by a persistent switch to rapid forward movement as animals seek to avoid and escape hypoxia. While the behavioral responses to 1% O₂ closely resemble those evoked by 21% O₂, they have distinct molecular and circuit underpinnings. Disrupting phosphodiesterases (PDEs), specific G proteins, or BBSome function inhibits escape from 1% O₂ due to increased cGMP signaling. A primary source of cGMP is GCY-28, the ortholog of the atrial natriuretic peptide (ANP) receptor. cGMP activates the protein kinase G EGL-4 and enhances neuroendocrine secretion to inhibit acute responses to 1% O₂. Triggering a rise in cGMP optogenetically in multiple neurons, including AIA interneurons, rapidly and reversibly inhibits escape from 1% O₂. Ca²⁺ imaging reveals that a 7% to 1% O₂ stimulus evokes a Ca²⁺ decrease in several neurons. Defects in mitochondrial complex I (MCI) and mitochondrial complex I (MCIII), which lead to persistently high reactive oxygen species (ROS), abrogate acute hypoxia responses. In particular, repressing the expression of isp-1, which encodes the iron sulfur protein of MCIII, inhibits escape from 1% O₂ without affecting responses to 21% O₂. Both genetic and pharmacological up-regulation of mitochondrial ROS increase cGMP levels, which contribute to the reduced hypoxia responses. Our results implicate ROS and precise regulation of intracellular cGMP in the modulation of acute responses to hypoxia by C. elegans.

The ability to detect and respond to acute oxygen shortages is indispensable to aerobic life, but the molecular mechanisms underlying this capacity are poorly understood. This study reveals that high levels of cGMP and reactive oxygen species (ROS) prevent the nematode Caenorhabditis elegans from escaping hypoxia.     

Sensory signaling-dependent remodeling of olfactory cilia architecture in C. elegans

Nonmotile primary cilia are sensory organelles composed of a microtubular axoneme and a surrounding membrane sheath that houses signaling molecules. Optimal cellular function requires the precise regulation of axoneme assembly, membrane biogenesis, and signaling protein targeting and localization via as yet poorly understood mechanisms. Here, we show that sensory signaling is required to maintain the architecture of the specialized AWB olfactory neuron cilia in C. elegans. Decreased sensory signaling results in alteration of axoneme length and expansion of a membraneous structure, thereby altering the topological distribution of a subset of ciliary transmembrane signaling molecules. Signaling-regulated alteration of ciliary structures can be bypassed by modulation of intracellular cGMP or calcium levels and requires kinesin-II-driven intraflagellar transport (IFT), as well as BBS- and RAB8-related proteins. Our results suggest that compensatory mechanisms in response to altered levels of sensory activity modulate AWB cilia architecture, revealing remarkable plasticity in the regulation of cilia structure.     

A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 control the timing of specific postembryonic developmental events in C. elegans. The experiments described here examine how these four genes interact to control a particular stage-specific event of the lateral hypodermal cell lineages. This event, termed the "larva-to-adult switch" (L/A switch), involves several coordinate changes in the behavior of hypodermal cells at the fourth molt: cessation of cell division, formation of adult (instead of larval) cuticle, cell fusion, and cessation of the molting cycle. The phenotypes of multiply mutant strains suggest a model wherein the L/A switch is controlled by the stage-specific activity of a regulatory hierarchy: At early stages of wild-type development, lin-14 and lin-28 inhibit lin-29 and thus prevent switching. Later, lin-4 inhibits lin-14 and lin-28, allowing activation of lin-29, which in turn triggers the switch in the L4 stage. lin-29 may activate the L/A switch by regulating genes that control cell division, differentiation, and stage-specific gene expression in hypodermal cells.     

ARK-1 inhibits EGFR signaling in C. elegans

A screen for synthetic enhancers of sli-1 identified ark-1 (forAck-related tyrosine kinase), a novel inhibitor of let-23 EGFR signaling in C. elegans. An ark-1 mutation synergizes with mutations in other negative regulators of let-23, resulting in increased RAS signaling. Genetic analysis suggests that ARK-1 acts upstream of RAS and is dependent upon SEM-5. ARK-1 inhibits LET-23-mediated ovulation, a RAS-independent function. ARK-1 physically interacts with SEM-5 in the yeast two-hybrid assay. We find that sem-5 also has a negative function in let-23-mediated ovulation and suggest that this negative function is mediated by the recruitment of inhibitors such as ARK-1.     

A gene expression fingerprint of C. elegans embryonic motor neurons

Background

ADP-ribosylation is an enzyme-catalyzed posttranslational protein modification in which mono(ADP-ribosyl)transferases (mARTs) and poly(ADP-ribosyl)transferases (pARTs) transfer the ADP-ribose moiety from NAD onto specific amino acid side chains and/or ADP-ribose units on target proteins.

Results

Using a combination of database search tools we identified the genes encoding recognizable pART domains in the public genome databases. In humans, the pART family encompasses 17 members. For 16 of these genes, an orthologue exists also in the mouse, rat, and pufferfish. Based on the degree of amino acid sequence similarity in the catalytic domain, conserved intron positions, and fused protein domains, pARTs can be divided into five major subgroups. All six members of groups 1 and 2 contain the H-Y-E trias of amino acid residues found also in the active sites of Diphtheria toxin and Pseudomonas exotoxin A, while the eleven members of groups 3 - 5 carry variations of this motif. The pART catalytic domain is found associated in Lego-like fashion with a variety of domains, including nucleic acid-binding, protein-protein interaction, and ubiquitylation domains. Some of these domain associations appear to be very ancient since they are observed also in insects, fungi, amoebae, and plants. The recently completed genome of the pufferfish T. nigroviridis contains recognizable orthologues for all pARTs except for pART7. The nearly completed albeit still fragmentary chicken genome contains recognizable orthologues for twelve pARTs. Simpler eucaryotes generally contain fewer pARTs: two in the fly D. melanogaster, three each in the mosquito A. gambiae, the nematode C. elegans, and the ascomycete microfungus G. zeae, six in the amoeba E. histolytica, nine in the slime mold D. discoideum, and ten in the cress plant A. thaliana. GenBank contains two pART homologues from the large double stranded DNA viruses Chilo iridescent virus and Bacteriophage Aeh1 and only a single entry (from V. cholerae) showing recognizable homology to the pART-like catalytic domains of Diphtheria toxin and Pseudomonas exotoxin A.

Conclusion

The pART family, which encompasses 17 members in the human and 16 members in the mouse, can be divided into five subgroups on the basis of sequence similarity, phylogeny, conserved intron positions, and patterns of genetically fused protein domains.     

Two sensory neurons coordinate the systemic mitochondrial stress response via GPCR signaling in C. elegans

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Odor recognition and second messenger signaling in olfactory receptor neurons

The detection of volatile odorants is supposed to begin with their interaction with soluble binding proteins which shuttle the hydrophobic ligands through the aqueous mucus layer towards specific odorant receptors in the ciliary membrane of olfactory neurons. A large family of receptors for odorants has been identified recently; individual receptor types are expressed in subsets of cells distributed in distinct zones of the olfactory epithelium. Ligand-receptor interaction triggers a rapid multistep reaction cascade, ultimately leading to an electrical response of the receptor neuron. Olfactory signaling is terminated by phosphorylation of receptors via a negative feedback reaction catalyzed by two types of kinases.     

Organogenesis in C. elegans: positioning of neurons and muscles in the egg-laying system

One of the final stages in the development of egg-laying behavior in the nematode C. elegans is the organization of 8 motor neurons (2 HSN and 6 VC cells) and 8 muscles into a motor system to control the opening of the vulva. Using mutations that disrupt the development of specific components of the egg-laying system and laser microsurgery to ablate selected precursor cells, we have determined that the guidance of the egg-laying neurons and muscles, and in particular the VC neurons and vulval muscles, into the vulval region is dependent on interactions with surrounding epithelial and gonadal tissue and appears to be independent of neuron-neuron and neuron-muscle interactions. The development of the egg-laying system can be described as a series of cell interactions in which certain cells arise through induction and subsequently provide inductive cues themselves.     

The long and the short of Wnt signaling in C. elegans

The simplicity of C. elegans makes it an outstanding system to study the role of Wnt signaling in development. Many asymmetric cell divisions in C. elegans require the Wnt/beta-catenin asymmetry pathway. Recent studies confirm that SYS-1 is a structurally and functionally divergent beta-catenin, and implicate lipids and retrograde trafficking in maintenance of WRM-1/beta-catenin asymmetry. Wnts also regulate short-range events such as spindle rotation and gastrulation, and a PCP-like pathway regulates asymmetric divisions. Long-range, cell non-autonomous Wnt signals regulate vulval induction. Both short-range and long-range Wnt signal s are regulated by recycling of MIG-14/Wntless via the retromer complex. These studies indicate that C. elegans continues to be useful for identifying new, conserved mechanisms underlying Wnt signaling in metazoans.